Recently new advances have been made in our understanding of the functional roles which nicotinic acetylcholine receptors may play in the brain, and the cloning of the genes for these receptors has given us the tools to study the detailed molecular mechanism of these receptors [l]. Nicotinic receptors are known to be involved in addictive processes[2], and have been suggested to be affected in schizophrenia [3, 4]. They have also been shown to be important for cognitive processes and memory. Nicotinic agonists are being developed as therapeutics for the treatment of Alzheimer's dementia [5]. Along with an increased understanding of the functional roles that nicotinic receptors may play in brain function has come an appreciation for the multiple receptor subtypes that exist in the brain. This proposal will extend our understanding of how a crucial physiological property, the permeability to divalent ions, is regulated both on the level of receptor subtype (i.e. subunit combination) and also in terms of the specific protein domains. Divalent ion permeability may be required for the neuronal plasticity associated with learning and memory and may also create a potential for excitotoxicity. We will express cloned nicotinic receptors in Xenopus oocytes, and with the study of chimeric and mutant subunits, we will identify the molecular elements that regulate calcium permeability, as well as the molecular elements that are involved with use-dependent inhibition of the receptors. By evaluating the relationships between the elements that regulate divalent ion permeability, and those which are associated with sensitivity to specific antagonists it may be possible to define therapeutics which may target specific functionally important receptor subtypes, either to spare those receptors important for cognitive processes, while targeting those involved in addictive processes, or to selectively block those which may put cells at risk of toxicity. The analysis of receptor physiology and pharmacology will be carried out both at the level of whole-cell currents and in terms of a detailed study of single channel properties. The study of a related series of bifunctional inhibitors will permit the disposition of inhibitory binding sites within the receptor complex to be evaluated. Our extensive preliminary data has provided us with a strong basis for experimental design in terms of approaches and candidate sequences for important structural domains. We have evidence that divalent ion permeability is regulated by the gamma subunits of muscle receptors and the alpha5 subunits of neuronal receptors. This model will be directly evaluated with antisense knockout experiments directed at eliminating the functional influences of these subunits in native receptors. The experiments in this proposal will provide important new insights into the nicotinic receptors of the brain in terms of their biophysical properties, their potential as therapeutic targets, and relationships between specific functional properties and drug sensitivity.